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1.
Reprod Fertil Dev ; 36(2): 112-123, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38064192

RESUMO

CONTEXT: Genome editing enables the introduction of beneficial sequence variants into the genomes of animals with high genetic merit in a single generation. This can be achieved by introducing variants into primary cells followed by producing a live animal from these cells by somatic cell nuclear transfer cloning. The latter step is associated with low efficiencies and developmental problems due to incorrect reprogramming of the donor cells, causing animal welfare concerns. Direct editing of fertilised one-cell embryos could circumvent this issue and might better integrate with genetic improvement strategies implemented by the industry. METHODS: In vitro fertilised zygotes were injected with TALEN editors and repair template to introduce a known coat colour dilution mutation in the PMEL gene. Embryo biopsies of injected embryos were screened by polymerase chain reaction and sequencing for intended biallelic edits before transferring verified embryos into recipients for development to term. Calves were genotyped and their coats scanned with visible and hyperspectral cameras to assess thermal energy absorption. KEY RESULTS: Multiple non-mosaic calves with precision edited genotypes were produced, including calves from high genetic merit parents. Compared to controls, the edited calves showed a strong coat colour dilution which was associated with lower thermal energy absorbance. CONCLUSIONS: Although biopsy screening was not absolutely accurate, non-mosaic, precisely edited calves can be readily produced by embryo-mediated editing. The lighter coat colouring caused by the PMEL mutation can lower radiative heat gain which might help to reduce heat stress. IMPLICATIONS: The study validates putative causative sequence variants to rapidly adapt grazing cattle to changing environmental conditions.


Assuntos
Edição de Genes , Genoma , Animais , Bovinos , Genótipo , Embrião de Mamíferos , Mutação
2.
Front Genet ; 13: 925913, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35899192

RESUMO

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

3.
Vet Immunol Immunopathol ; 114(1-2): 135-48, 2006 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-16956667

RESUMO

The expression of interleukin (IL)-4, IL-5, IL-10, IL-13, TNF-alpha and IFN-gamma genes, and parasite-specific IgM, IgG1, IgG2, IgA and total IgE levels, were monitored daily in intestinal lymph of sheep infected repeatedly with the nematode parasite Trichostrongylus colubriformis. Host genotype had a significant influence on IL-13 gene activity, with resistant-line (R) sheep consistently expressing higher levels of mRNA than susceptible-line (S) sheep. Mean gene expression of IL-13, IL-4 and IFN-gamma did not differ significantly between the first and second nematode challenge. Field-primed R and S as well as field-primed R and naïve S sheep had lower mean gene expression of IL-5 and IL-10, respectively, during the second when compared to primary challenge. Genes for IL-13 and IL-5 were transiently and strongly up-regulated after nematode infection, particularly in animals with previous exposure to nematodes. Genes for TNF-alpha and IFN-gamma were also transiently up-regulated, but to a lesser extent and more typically after primary challenge. Naïve sheep of both genotypes produced relatively little antibody response after primary challenge. A second nematode challenge resulted in large increases in the lymphatic levels of all antibody sub-classes which were significant for adult antigen-specific IgA and larval antigen-specific IgG1. In naïve S line sheep, the larval-specific IgA and IgG2 response appeared delayed when compared to the R line animals. Field-primed R and S line sheep had relatively high lymphatic IgG1 levels prior to experimental infection and these did not change significantly afterwards. These results demonstrate that during nematode infections, the intestinal micro-environment of sheep is transiently skewed towards Th2 cytokine dominance, although IFN-gamma gene expression continues. This response is accompanied by increases of nematode-specific IgG1, IgA, IgG2 and IgM, as well as of total IgE in lymph plasma.


Assuntos
Citocinas/imunologia , Enteropatias Parasitárias/veterinária , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Ovinos/imunologia , Tricostrongilose/veterinária , Trichostrongylus/imunologia , Animais , Anticorpos Anti-Helmínticos/biossíntese , Anticorpos Anti-Helmínticos/imunologia , Citocinas/genética , Fezes/parasitologia , Regulação da Expressão Gênica/imunologia , Isotipos de Imunoglobulinas/biossíntese , Isotipos de Imunoglobulinas/imunologia , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/imunologia , Enteropatias Parasitárias/parasitologia , Linfa/imunologia , Linfa/parasitologia , Masculino , Contagem de Ovos de Parasitas/veterinária , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Ovinos/genética , Estatísticas não Paramétricas , Tricostrongilose/genética , Tricostrongilose/imunologia , Tricostrongilose/parasitologia
4.
Infect Immun ; 73(4): 2175-83, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15784560

RESUMO

Cytokine gene expression in cells migrating in afferent and efferent intestinal lymph was monitored for extended time periods in individual sheep experimentally infected with the nematode Trichostrongylus colubriformis. Animals from stable selection lines with increased levels of either genetic resistance (R) or susceptibility (S) to nematode infection were used. Genes for interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha (TNF-alpha), but not for IL-4, IL-10, or gamma interferon (IFN-gamma), were consistently expressed at higher levels in both afferent and efferent lymph cells of R sheep than in S sheep. However, only minor differences were observed in the surface phenotypes and antigenic and mitogenic responsiveness of cells in intestinal lymph between animals from the two selection lines. The IL-4 and IL-10 genes were expressed at higher levels in afferent lymph cells than in efferent lymph cells throughout the course of the nematode infection in animals of both genotypes, while the proinflammatory TNF-alpha gene was relatively highly expressed in both lymph types. These relationships notwithstanding, expression of the IL-10 and TNF-alpha genes declined significantly in afferent lymph cells but not in efferent lymph cells during infection. Collectively, the results showed that R-line sheep developed a strong polarization toward a Th2-type cytokine profile in immune cells migrating in lymph from sites where the immune response to nematodes was initiated, although the IFN-gamma gene was also expressed at moderate levels. Genes or alleles that predispose an animal to develop this type of response appear to have segregated with the R selection line and may contribute to the increased resistance of these animals.


Assuntos
Interleucina-13/genética , Interleucina-5/genética , Intestinos/imunologia , Linfa/imunologia , Tricostrongilose/imunologia , Trichostrongylus/imunologia , Fator de Necrose Tumoral alfa/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Genótipo , Interferon gama/genética , Interleucina-10/genética , Interleucina-4/genética , Intestinos/parasitologia , Linfa/citologia , Masculino , Ovinos , Tricostrongilose/parasitologia
5.
J Immunol Methods ; 293(1-2): 153-68, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15541285

RESUMO

Reliable methods for long-term collection of afferent lymph draining from the small intestine of sheep are described and validated. The procedure was used successfully in normal sheep, in animals infected experimentally with the parasitic intestinal nematode Trichostrongylus colubriformis and in animals infected naturally with Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. Our approach enabled afferent lymph draining from the small intestine to be collected continuously for up to 4 months, without any detrimental effects on the animals. Based on cytokine gene expression profiles of afferent intestinal lymph cells, the two infections induced contrasting regional immune responses, namely, Th2-type immunity in the case of T. colubriformis infection and Th1-type immunity in natural cases of Johne's disease. Some immune parameters differed markedly between the two disease models, highlighting the potential value of this approach to gain real-time insights into distinctive host-pathogen interactions as they occur in vivo within the regional immune system of the gastrointestinal tract.


Assuntos
Intestino Delgado/fisiologia , Linfa/fisiologia , Ovinos/fisiologia , Animais , Intestino Delgado/imunologia , Intestino Delgado/cirurgia , Linfa/imunologia , Vasos Linfáticos/cirurgia , Mycobacterium avium/imunologia , Ovinos/imunologia , Ovinos/microbiologia , Ovinos/parasitologia , Tricostrongilose/imunologia , Tuberculose/imunologia , Tuberculose/veterinária
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